Complex chromosome engineering for production of human antibodies in transgenic animals

Abstract

The invention relates to large-scale production of human antibodies by transgenic animals with high production of fully human IgG up to >10 g/L in sera with human IgG1 subclass dominancy. This invention also supports a feasibility of complex chromosome engineering for complicated genetic studies in non-murine mammalian species.

The invention claimed is: 
     
       1. A human artificial chromosome (HAC) vector comprising one or more human immunoglobulin heavy and light chain loci that are capable of undergoing rearrangement and expression in B-cells to produce a human immunoglobulin in response to exposure to one or more antigens, wherein said immunoglobulin loci comprise:
 (a) a gene encoding one or more human antibody heavy chains comprising at least a human IgG heavy chain, a human IgA heavy chain or both human IgG and IgA heavy chains, wherein each gene encoding an antibody heavy chain is operatively linked to its own class switch regulatory element upstream of the human antibody heavy chain; 
 (b) a gene encoding one or more human antibody light chains; and 
 (c) a gene encoding a human-ungulate chimeric IgM heavy chain constant region encoding constant heavy (CH) domains CH1, CH2, CH3 and CH4, and transmembrane domains (TM) TM1 and TM2, wherein the encoded CH1, CH2, and CH3 domains are human, and the encoded CH4, TM1 and TM2 domains are ungulate; 
 wherein at least one class switch regulatory element of the genes encoding the one or more human antibody heavy chains is replaced with an ungulate class switch regulatory element selected from the group consisting of Imu-Smu (Iμ-Sμ), Igamma-Sgamma (Iγ-Sγ), Ialpha-Salpha (Iα-Sα), and Iepsilon-Sepsilon (Iε-Sε) ungulate class switch regulatory elements. 
 
     
     
       2. The HAC of  claim 1 , wherein the gene encoding the one or more human antibody heavy chains is a human IgG antibody heavy chain. 
     
     
       3. The HAC vector of  claim 2 , wherein the IgG heavy chain is an IgG1 antibody heavy chain. 
     
     
       4. The HAC vector of  claim 1 , wherein the gene encoding the one or more human antibody heavy chains is a human IgA antibody heavy chain. 
     
     
       5. The HAC vector of  claim 1 , wherein the gene encoding the one or more human antibody heavy chains further comprises a human IgM antibody heavy chain. 
     
     
       6. The HAC vector of  claim 1 , wherein the one or more human antibody heavy chains is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. 
     
     
       7. The HAC vector of  claim 1 , wherein the gene encoding the human-ungulate chimeric IgM heavy chain constant region is further modified so that:
 (a) the encoded CH1 domain is a human-ungulate chimeric CH1 domain, and the encoded CH2, CH3, CH4, and TM1 and TM2 domains are ungulate; or 
 (b) the encoded CH1 domain is human, the encoded CH2 domain is a human-ungulate chimeric CH2 domain, and the encoded CH3, CH4, and TM1 and TM2 domains are ungulate. 
 
     
     
       8. The HAC vector of  claim 7 , wherein the encoded CH2, CH3, CH4, and TM1 and TM2 ungulate domains are bovine, the human-ungulate chimeric CH1 domain is a human-bovine chimeric CH1 domain and the human-ungulate chimeric CH2 domain is a human-bovine chimeric CH2 domain. 
     
     
       9. The HAC of  claim 1 , wherein the one or more human antibody heavy chains comprises a human IgG antibody heavy chain, wherein a transmembrane domain and an intracellular domain of a constant region of the human IgG heavy antibody chain are replaced with a transmembrane domain and an intracellular domain of an ungulate IgG antibody heavy chain constant region. 
     
     
       10. The HAC vector of  claim 9 , wherein the human IgG antibody heavy chain is a human IgG1 antibody heavy chain. 
     
     
       11. The HAC vector of  claim 9 , wherein the ungulate IgG antibody heavy chain constant region is a bovine IgG antibody heavy chain constant region. 
     
     
       12. The HAC vector of  claim 1 , wherein the ungulate class switch regulatory element is an Igamma-Sgamma (Iγ-Sγ) class switch regulatory element. 
     
     
       13. The HAC vector of  claim 12 , wherein the Iγ-Sγ class switch regulatory element is an Igamma1-Sgamma1 (I γ1 -S γ1 ). 
     
     
       14. The HAC vector of  claim 1 , wherein each class switch regulatory element of the genes encoding the one or more human antibody heavy chains is replaced with an ungulate class switch regulatory element. 
     
     
       15. The HAC vector of  claim 1 , wherein the ungulate class switch regulatory element(s) are bovine class switch regulatory elements. 
     
     
       16. The HAC vector of  claim 1 , wherein the HAC vector comprises one or more genes encoding a human antibody surrogate light chain selected from the group consisting of pre-B lymphocyte 1 (VpreB1), pre-B lymphocyte 3(VpreB3) and lambda 5 (λ5) human antibody surrogate light chains. 
     
     
       17. The HAC vector of  claim 1 , further comprising an ungulate enhancer operatively linked to one or more genes encoding the one or more human antibody heavy chains. 
     
     
       18. The HAC vector of  claim 17 , wherein the enhancer is a 3′enhancer alpha (3′Ea enhancer). 
     
     
       19. A transgenic ungulate comprising the HAC vector of  claim 1 . 
     
     
       20. The transgenic ungulate of  claim 19 , wherein the transgenic ungulate is a transgenic bovine. 
     
     
       21. The HAC vector of  claim 1 , wherein the gene encoding the one or more human antibody light chains encodes at least a human kappa (κ) light chain, a human lambda (λ) light chain or both human kappa and lambda light chains. 
     
     
       22. The HAC vector of  claim 6 , further comprising one or more human antibody IgE and IgD human antibody heavy chains.